Drug Discovery
Drug discovery relies on the ability to identify compounds that interact with a selected target, such as a cell, an antibody, receptor, enzyme, transcription factor or the like. Traditional drug discovery relied on collections or "libraries" obtained from proprietary databases of compounds accumulated over many years, natural products, fermentation broths, and rational drug design. Recent advances in molecular biology, chemistry and automation have resulted in the development of rapid, high throughput screening (HTS) protocols to screen these collections. In connection with HTS, methods for generating molecular diversity and for detecting, identifying and quantifying biological or chemical material have been developed. These advances have been facilitated by fundamental developments in chemistry, including the development of highly sensitive analytical methods, solid state chemical synthesis, and sensitive and specific biological assay systems.
Analyses of biological interactions and chemical reactions, however, require the use of labels or tags to track and identify the results of such analyses. Typically biological reactions, such as binding, catalytic, hybridization and signaling reactions, are monitored by labels, such as radioactive, fluorescent, photoabsorptive, luminescent and other such labels, or by direct or indirect enzyme labels. Chemical reactions are also monitored by direct or indirect means, such as by linking the reactions to a second reaction in which a colored, fluorescent, chemoluminescent or other such product results. These analytical methods, however, are often time consuming, tedious and, when practiced in vivo, invasive. In addition, each reaction is typically measured individually, in a separate assay. There is, thus, a need to develop alternative and convenient methods for tracking and identifying analytes in biological interactions and the reactants and products of chemical reactions.
Combinatorial Libraries
The provision and maintenance of compounds to support HTS have become critical. New methods for the lead generation and lead optimization have emerged to address this need for diversity. Among these methods is combinatorial chemistry, which has become a powerful tool in drug discovery and materials science. Methods and strategies for generating diverse libraries, primarily. peptide- and nucleotide-based oligomer libraries, have been developed using molecular biology methods and/or simultaneous chemical synthesis methodologies (see, e.g., A Practical Guide to Combinatorial Chemistry, DeWitt, S. H. (1997) Czarnik, A. W., Editors, ACS Books, Washington; Combinatorial Chemistry: Synthesis and Application, Wilson, S. H. (1997) Czarnik, A. W., Editors, Wiley & Sons, NY, N.Y.; Dower et al. (1991) Annu. Rep. Med. Chem. 26:271-280; Fodor et al. (1991) Science 251:767-773; Jung et al. (1992) Angew. Chem. Ind. Ed. Engl. 31:367-383; Zuckerman et al. (1992) Proc. Natl. Acad. Sci. USA 89:4505-4509; Scott et al. (1990) Science 249:386-390; Devlin et al. (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and Gallop et al. (1994) J. Medicinal Chemistry 37:1233-1251). The resulting combinatorial libraries potentially contain millions of pharmaceutically relevant compounds and that can be screened to identify compounds that exhibit a selected activity.
High Throughput Screening
In addition, exploitation of this diversity requires development of methods for rapidly screening compounds. Advances in instrumentation, molecular biology and protein chemistry, and the adaptation of biochemical activity screens into microplate formats, has made it possible to screen of large numbers of compounds. Also, because compound screening has been successful in areas of significance for the pharmaceutical industry, high throughput screening (HTS) protocols have assumed importance. Presently, there are hundreds of HTS systems operating throughout the world, which are used, not only for compound screening for drug discovery, but also for immunoassays, cell-based assays and receptor-binding assays.
An essential element of high throughput screening for drug discovery process and areas in which molecules are identified and tracked, is the ability to extract the information made available during synthesis and screening of a library, identification of the active components of intermediary structures, and the reactants and products of assays. While there are several techniques for identification of intermediary products and final products, nanosequencing protocols that provide exact structures are only applicable on mass to naturally occurring linear oligomers such as peptides and amino acids. Mass spectrographic (MS) analysis is sufficiently sensitive to determine the exact mass and fragmentation patterns of individual synthesis steps, but complex analytical mass spectrographic strategies are not readily automated nor conveniently performed. Also, mass spectrographic analysis provides at best simple connectivity information, but no stereoisomeric information, and generally cannot discriminate among isomeric monomers. Another problem with mass spectrographic analysis is that it requires pure compounds; structural determinations on complex mixtures is either difficult or impossible. Finally, mass spectrographic analysis is tedious and time consuming. Thus, although there are a multitude of solutions to the generation of libraries and to screening protocols, there are no ideal solutions to the problems of identification, tracking and categorization.
These problems arise in any screening or analytical process in which large numbers of molecules or biological entities are screened. In any system, once a desired molecule(s) has been isolated, it must be identified. Simple means for identification do not exist. Because of the problems inherent in any labeling procedure, it would be desirable to have alternative means for tracking and quantitating chemical and biological reactions during synthesis and/or screening processes, and for automating such tracking and quantitating.
Solid Supports
A key feature in the use of combinatorial chemistry and high throughput screening in drug discovery is the solid support used during synthesis of the libraries. Such supports take a variety of forms, including, but not limited to, inorganics, natural polymers, and synthetic polymers, including, but not limited to: cellulose, cellulose derivatives, acrylic resins, glass that is derivatized to render it suitable for use a support, silica gels, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, polystyrene cross-linked with divinylbenzene or the like (see, Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamides, latex gels, dextran, rubber, silicon, plastics, nitrocellulose, natural sponges, metals, plastic, cross-linked dextrans, such as those sold under the tradename Sephadex (Pharmacia) and agarose gel, such as gels sold under the tradename Sepharose (Pharmacia), which is a hydrogen bonded polysaccharide-type agarose gel.
Radiation grafting of monomers allows a diversity of surface characteristics to be generated on polymeric supports (see, e.g., Maeji et al. (1994) Reactive Polymers 22:203-212; and Berg et al. (1989) J. Am. Chem. Soc. 111:8024-8026). For example, radiolytic grafting of monomers, such as vinyl monomers, or mixtures of monomers, to polymers, such as polyethylene and polypropylene, produce composites that have a wide variety of surface characteristics. These methods have been used to graft polymers to insoluble supports, particularly polypropylene, for synthesis of peptides and other molecules. These methods have not been successfully employed for fluoropolymers.
It is important for the supports to be resistant to the conditions in which syntheses and/or assays are performed. Consequently, fluoropolymeric materials for use as resins and solid supports, which are highly inert materials that are resistant to solvents and temperatures employed during synthesis would be widely employed in combinatorial chemistry. The disadvantage in using these polymers, however, is the difficulty encountered in binding or covalently bonding a substrate of interest because of their inert character. In combinatorial synthesis, the solid supports generally must possess functionality or be derivatized in such a way as to be able to covalently or otherwise bind a substrate of interest during the combinatorial synthesis. Typical functional groups include alcohols, amines, alkyl halides, phenols, aldehydes, nitriles, carboxyl groups and the like. Thus, in order to use highly inert fluoropolymeric resins as solid supports in combinatorial chemistry, the resins must be derivatized to allow for binding of a substrate of interest. The methods available for grafting polymers to fluoropolymers yield fluoropolymers in which the copolymer level of grafting is not sufficient to render the resulting surfaces suitable for use in synthesis and/or screening assays.
Therefore, it is an object herein to provide methods for irradiation induced graft polymerization that provide graft polymers of sufficiently high level of grafting such that the resulting grafted polymer (composite material) is suitable for use as a support in syntheses and screening, particularly for in combinatorial synthetic and high throughput screening protocols. It is also an object herein to provide the graft copolymers produced by the methods.